Grand Fir / Snowbrush Microbiome

2024-01-31

Preliminary data exploration

Contaminant ASVs were identified with the prevalence method using the decontam package.

Plant measures overview

Scaled-centered plant ‘health’ measures, broken down by treatment factors.

Model output

Formula: value ~ drought * host * fire_freq + (1|inoculum_site/block))
effect term estimate std.error statistic df p.value
fixed (Intercept) 0.071 0.280 0.255 5.343 0.808
fixed droughtND -0.017 0.163 -0.106 384.262 0.916
fixed hostCeanothus velutinus -0.522 0.164 -3.190 381.647 0.002
fixed fire_freq 0.104 0.154 0.675 5.360 0.528
fixed droughtND:hostCeanothus velutinus 0.560 0.232 2.412 383.262 0.016
fixed droughtND:fire_freq -0.037 0.089 -0.416 381.323 0.677
fixed hostCeanothus velutinus:fire_freq -0.002 0.090 -0.017 382.464 0.987
fixed droughtND:hostCeanothus velutinus:fire_freq -0.033 0.127 -0.263 382.083 0.793



Inoculum communities overview

Taxonomic overview

Bacteria

Alpha diversity

Effect of Shannon diversity on plant measures

effect group term estimate std.error statistic df p.value
fixed NA (Intercept) -0.733 0.476 -1.541 360.365 0.124
fixed NA Shannon 0.129 0.085 1.514 664.977 0.130
fixed NA hostCeanothus velutinus 0.898 0.708 1.269 664.182 0.205
fixed NA droughtND 0.448 1.182 0.379 678.507 0.705
fixed NA fire_freq 1.654 0.563 2.938 668.475 0.003
fixed NA Shannon:hostCeanothus velutinus -0.163 0.125 -1.304 658.644 0.193
fixed NA Shannon:droughtND -0.088 0.212 -0.415 678.004 0.678
fixed NA hostCeanothus velutinus:droughtND 0.379 1.959 0.193 696.563 0.847
fixed NA Shannon:fire_freq -0.282 0.100 -2.824 677.140 0.005
fixed NA hostCeanothus velutinus:fire_freq -3.494 1.363 -2.564 681.500 0.011
fixed NA droughtND:fire_freq -1.120 0.838 -1.336 673.807 0.182
fixed NA Shannon:hostCeanothus velutinus:droughtND -0.070 0.344 -0.203 696.859 0.839
fixed NA Shannon:hostCeanothus velutinus:fire_freq 0.597 0.221 2.696 683.713 0.007
fixed NA Shannon:droughtND:fire_freq 0.196 0.151 1.301 672.746 0.194
fixed NA hostCeanothus velutinus:droughtND:fire_freq 3.048 1.773 1.719 687.380 0.086
fixed NA Shannon:hostCeanothus velutinus:droughtND:fire_freq -0.521 0.293 -1.777 690.349 0.076
ran_pars inoculum_site:block sd__(Intercept) 0.296 NA NA NA NA
ran_pars block sd__(Intercept) 0.332 NA NA NA NA
ran_pars Residual sd__Observation 0.895 NA NA NA NA

Fungi

Alpha diversity

Effect of Shannon diversity on plant measures

effect group term estimate std.error statistic df p.value
fixed NA (Intercept) 0.123 0.274 0.448 71.682 0.656
fixed NA Shannon -0.172 0.181 -0.954 621.589 0.341
fixed NA hostCeanothus velutinus 0.192 0.513 0.374 612.037 0.708
fixed NA droughtND 0.203 0.310 0.654 697.861 0.513
fixed NA fire_freq 0.200 0.198 1.010 526.205 0.313
fixed NA Shannon:hostCeanothus velutinus -0.049 0.340 -0.144 582.109 0.886
fixed NA Shannon:droughtND -0.251 0.275 -0.911 701.406 0.363
fixed NA hostCeanothus velutinus:droughtND -0.492 0.609 -0.807 676.824 0.420
fixed NA Shannon:fire_freq -0.087 0.152 -0.571 613.249 0.568
fixed NA hostCeanothus velutinus:fire_freq -0.178 0.420 -0.425 635.728 0.671
fixed NA droughtND:fire_freq -0.215 0.230 -0.933 689.863 0.351
fixed NA Shannon:hostCeanothus velutinus:droughtND 0.387 0.439 0.881 681.222 0.379
fixed NA Shannon:hostCeanothus velutinus:fire_freq 0.159 0.244 0.652 623.003 0.515
fixed NA Shannon:droughtND:fire_freq 0.187 0.192 0.974 687.152 0.330
fixed NA hostCeanothus velutinus:droughtND:fire_freq 0.217 0.472 0.459 675.089 0.646
fixed NA Shannon:hostCeanothus velutinus:droughtND:fire_freq -0.285 0.304 -0.940 682.735 0.348
ran_pars inoculum_site:block sd__(Intercept) 0.264 NA NA NA NA
ran_pars block sd__(Intercept) 0.334 NA NA NA NA
ran_pars Residual sd__Observation 0.901 NA NA NA NA

Comparison between inoculum and root communities

Bacteria

Not all taxa in the inoculae were subsequently found in the root samples

Fungi




Microbial guilds

Bacteria

I couldn’t get a trustworthy annotation of “mutualists” for bacteria, but here are probable pathogens:

effect term estimate std.error statistic df p.value species
fixed (Intercept) -0.8572 0.2935 -2.9206 49.6839 0.0052 Ceanothus
fixed proportion_pathogen 1.5096 0.6362 2.3727 410.9860 0.0181 Ceanothus
fixed droughtND 1.1837 0.3866 3.0617 408.2273 0.0023 Ceanothus
fixed fire_freq 0.2444 0.1865 1.3108 407.9500 0.1907 Ceanothus
fixed proportion_pathogen:droughtND -2.0673 1.1062 -1.8689 409.0361 0.0624 Ceanothus
fixed proportion_pathogen:fire_freq -0.2759 0.5126 -0.5383 408.5644 0.5906 Ceanothus
fixed droughtND:fire_freq 0.0513 0.2604 0.1968 409.6223 0.8441 Ceanothus
fixed proportion_pathogen:droughtND:fire_freq -0.5892 0.7795 -0.7558 410.0850 0.4502 Ceanothus
fixed (Intercept) -0.6413 0.4190 -1.5306 66.8098 0.1306 Abies
fixed proportion_pathogen 1.8993 1.1867 1.6005 487.9044 0.1101 Abies
fixed droughtND 0.9255 0.4772 1.9394 489.1544 0.0530 Abies
fixed fire_freq 0.3495 0.1975 1.7700 486.3527 0.0773 Abies
fixed proportion_pathogen:droughtND -2.8146 1.4874 -1.8923 488.8933 0.0590 Abies
fixed proportion_pathogen:fire_freq -0.8519 0.7193 -1.1845 486.3072 0.2368 Abies
fixed droughtND:fire_freq -0.4264 0.2898 -1.4712 486.9375 0.1419 Abies
fixed proportion_pathogen:droughtND:fire_freq 1.1320 0.9384 1.2063 486.9307 0.2283 Abies

Fungi

How does the proportion of mutualist fungi relate to indicators of plant health?

In the following figure, the X-axis represents the proportion of fungi that could be identified to a mutualistic guild; the Y-axis represents the value of the various measures of plant success (facets). Y-axis scales vary between facets.

We see an opposite pattern when looking at the proportions of putatively pathogenic fungi

effect term estimate std.error statistic df p.value species
fixed (Intercept) -0.1914 0.1861 -1.0288 12.3567 0.3233 Ceanothus
fixed proportion_pathogen -0.8657 0.4922 -1.7586 407.2118 0.0794 Ceanothus
fixed droughtND 0.4250 0.1620 2.6238 407.5730 0.0090 Ceanothus
fixed fire_freq 0.1795 0.0826 2.1721 408.1123 0.0304 Ceanothus
fixed proportion_pathogen:droughtND -0.0517 0.7572 -0.0683 410.3794 0.9456 Ceanothus
fixed proportion_pathogen:fire_freq -0.0294 0.2703 -0.1087 408.7904 0.9135 Ceanothus
fixed droughtND:fire_freq -0.1092 0.1029 -1.0610 408.9926 0.2893 Ceanothus
fixed proportion_pathogen:droughtND:fire_freq -0.5012 0.5492 -0.9126 410.9484 0.3620 Ceanothus
fixed (Intercept) -0.0252 0.2310 -0.1090 7.9855 0.9159 Abies
fixed proportion_pathogen -0.8837 0.5533 -1.5972 487.2401 0.1109 Abies
fixed droughtND 0.1040 0.1283 0.8108 487.2950 0.4179 Abies
fixed fire_freq 0.0964 0.0538 1.7910 484.7280 0.0739 Abies
fixed proportion_pathogen:droughtND 0.0805 0.6645 0.1212 487.6918 0.9036 Abies
fixed proportion_pathogen:fire_freq 0.2447 0.2306 1.0613 485.8624 0.2891 Abies
fixed droughtND:fire_freq -0.1060 0.0721 -1.4694 484.9722 0.1424 Abies
fixed proportion_pathogen:droughtND:fire_freq -0.0540 0.3846 -0.1404 485.4858 0.8884 Abies
fixed (Intercept) -0.3606 0.2168 -1.6636 17.0196 0.1145 Ceanothus
fixed proportion_mutualist 0.0227 0.2761 0.0822 408.2311 0.9345 Ceanothus
fixed droughtND 0.4699 0.2285 2.0564 407.7090 0.0404 Ceanothus
fixed fire_freq 0.1075 0.0744 1.4441 407.1394 0.1495 Ceanothus
fixed proportion_mutualist:droughtND -0.0395 0.4118 -0.0960 408.5606 0.9236 Ceanothus
fixed proportion_mutualist:fire_freq 0.2470 0.2528 0.9768 408.2959 0.3293 Ceanothus
fixed droughtND:fire_freq -0.1153 0.1154 -0.9991 407.9088 0.3184 Ceanothus
fixed proportion_mutualist:droughtND:fire_freq -0.1034 0.3093 -0.3345 409.0122 0.7382 Ceanothus
fixed (Intercept) -0.7047 0.2849 -2.4733 19.8113 0.0226 Abies
fixed proportion_mutualist 0.9018 0.2626 3.4342 487.8720 0.0006 Abies
fixed droughtND 0.4584 0.2768 1.6561 488.7631 0.0984 Abies
fixed fire_freq 0.3142 0.0919 3.4196 487.1292 0.0007 Abies
fixed proportion_mutualist:droughtND -0.6042 0.3647 -1.6569 489.4817 0.0982 Abies
fixed proportion_mutualist:fire_freq -0.2953 0.1455 -2.0295 487.4639 0.0429 Abies
fixed droughtND:fire_freq -0.3247 0.1468 -2.2124 488.1415 0.0274 Abies
fixed proportion_mutualist:droughtND:fire_freq 0.3414 0.2081 1.6404 488.5207 0.1016 Abies



Beta-Diversity

Bacteria

## PermANOVA run without outlier Sterile-inoculum root samples...
term df SumOfSqs R2 statistic p.value
host 1 3.8414120 0.0687480 11.063965 0.001
fire_freq 1 1.7434756 0.0312022 5.021527 0.001
drought 1 0.8088162 0.0144750 2.329538 0.001
host:fire_freq 1 0.7153777 0.0128028 2.060418 0.002
host:drought 1 0.7562988 0.0135351 2.178278 0.002
fire_freq:drought 1 0.3814427 0.0068265 1.098624 0.234
host:fire_freq:drought 1 0.4106606 0.0073494 1.182777 0.162
Residual 136 47.2192419 0.8450610 NA NA
Total 143 55.8767255 1.0000000 NA NA

Fungi

## PermANOVA run without outlier Sterile-inoculum root samples...
term df SumOfSqs R2 statistic p.value
host 1 1.9352113 0.0300393 4.6753844 0.001
fire_freq 1 3.4231420 0.0531356 8.2701587 0.001
drought 1 0.5712680 0.0088675 1.3801580 0.113
host:fire_freq 1 0.9705051 0.0150646 2.3446971 0.003
host:drought 1 0.3803731 0.0059043 0.9189645 0.583
fire_freq:drought 1 0.5128794 0.0079612 1.2390938 0.202
host:fire_freq:drought 1 0.3369443 0.0052302 0.8140424 0.746
Residual 136 56.2924276 0.8737973 NA NA
Total 143 64.4227507 1.0000000 NA NA



Important Taxa

These taxa were identified by both Random Forest models and corncob as being important predictors of the various experimental conditions & had significant differential abundance between levels of those variables

Bacteria

Differential Abundance

Relative Abundances of ‘important’ taxa in root samples

Fungi

Differential Abundance

No fungal taxa were found to have significant differential abundance between drought levels

Relative Abundances of ‘important’ taxa in root samples



Phylogenetic properties

Bacteria

  • Still kind of messy
  • What specific questions do we need to address with this?

Fungi

  • Phylogenetic dispersion is tricky with ITS markers :(
  • Could try GhostTree for fungi of interest that come up as ‘unknown’



Network properties

Selected network attributes

Network properties vs Plant Health

(Composite scaled-centered plant health measures)

net_mod$formula

term estimate std.error statistic p.value
(Intercept) 0.4218878 0.1634647 2.5809105 0.0099162
speciesSnowbrush 0.0392213 0.0644262 0.6087778 0.5427327
kingdomFungi 0.0612656 0.0624022 0.9817862 0.3263098
n_vertices -0.0048043 0.0021046 -2.2827358 0.0225380
n_edges 0.0010962 0.0005309 2.0647123 0.0390636
mean_dist 0.0025417 0.0387610 0.0655733 0.9477233
mean_betweenness 0.0000157 0.0013290 0.0118412 0.9905533
mean_closeness -0.2699799 0.1797738 -1.5017755 0.1332942
global_effic -0.3549984 0.1904798 -1.8637063 0.0624920
clustering_coeficient -0.0277149 0.0869359 -0.3187974 0.7499095
max_degree 0.0073817 0.0033499 2.2035514 0.0276560
speciesSnowbrush:kingdomFungi -0.1135960 0.0725576 -1.5655978 0.1175822
speciesSnowbrush:n_vertices -0.0000737 0.0012860 -0.0573301 0.9542873
kingdomFungi:n_vertices -0.0019574 0.0017069 -1.1467603 0.2516016
speciesSnowbrush:kingdomFungi:n_vertices 0.0017051 0.0017246 0.9887404 0.3228956


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Soil chemistry

Overview of soil properties for each inoculum source:

Testing for significant differences between the samples…

Here are the soil properties that differ significantly between inoculum sources. Sterile inoculum soils are set as the intercept. The stats below pretty nicely reflect the obvious patterns in the overall figure.
term estimate std.error statistic p.value
Mn 1.248 0.268 4.658 0.000
NH4 0.770 0.268 2.875 0.004
Fe:inoc_site1 1.516 0.328 4.618 0.000
K:inoc_site1 0.904 0.328 2.753 0.006
NO3:inoc_site1 -0.793 0.328 -2.417 0.016
TKN:inoc_site1 0.735 0.328 2.240 0.026
Zn:inoc_site1 1.160 0.328 3.536 0.000
Fe:inoc_site2 0.831 0.328 2.532 0.012
K:inoc_site2 0.801 0.328 2.440 0.015
Mn:inoc_site2 -1.081 0.328 -3.294 0.001
TKN:inoc_site2 0.660 0.328 2.010 0.045
Mn:inoc_site3 -1.526 0.328 -4.648 0.000
NH4:inoc_site3 -0.976 0.328 -2.973 0.003
Zn:inoc_site3 -0.693 0.328 -2.112 0.035
C:N:inoc_site4 -0.893 0.328 -2.721 0.007
EC:inoc_site4 -1.026 0.328 -3.127 0.002
Mn:inoc_site4 -1.349 0.328 -4.110 0.000
B:inoc_site5 1.369 0.328 4.171 0.000
Ca:inoc_site5 1.192 0.328 3.631 0.000
K:inoc_site5 0.683 0.328 2.080 0.038
Mg:inoc_site5 0.682 0.328 2.078 0.039
Mn:inoc_site5 -1.099 0.328 -3.350 0.001
NO3:inoc_site5 1.854 0.328 5.649 0.000
TKN:inoc_site5 1.022 0.328 3.114 0.002
Zn:inoc_site5 1.448 0.328 4.412 0.000
B:inoc_site6 0.918 0.328 2.798 0.005
Mn:inoc_site6 -1.535 0.328 -4.678 0.000
NH4:inoc_site6 -0.754 0.328 -2.299 0.022
NO3:inoc_site6 1.273 0.328 3.880 0.000
Zn:inoc_site6 1.290 0.328 3.932 0.000